Continuous Isocratic Affinity Chromatography

ABSTRACT

The present invention provides devices and methods for micro-scale simulated moving bed chromatography (SMB) for continuous preparation of analytic quantities of highly pure fractions of target molecules. The present apparatus and method of the invention is adapted in a preferred embodiment to separations by affinity chromatography involving three discontinuous liquid flow loops. An alternative embodiment of affinity chromatography utilizes standard SMB operating under isocratic conditions.

This application claims the benefit of U.S. Prov. Appl. 60/918,617,filed Mar. 16, 2007 and U.S. Prov. Appl. 60/841,296, filed Aug. 30,2007, both of which are incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

The present invention relates to small-scale simulated moving bedchromatography (SMB) for continuous preparation of highly pure fractionsof target molecules. The present apparatus and method of the inventionare adapted in an embodiment to separations by chromatography involvingdiscontinuous liquid flow loops. A preferred embodiment of affinitychromatography utilizes SMB operating under isocratic conditions.

BACKGROUND OF THE INVENTION

Simulated moving bed chromatography (SMB) was first described in U.S.Pat. No. 2,985,589. The specification discloses a separation towerdivided into a number of individual interconnecting separation bedscontaining solid phase chromatography substrates. An inline pump at thebottom of the tower connects flow from the bottom to the top, therebyproviding a continuous loop. Inlet ports for feedstock (F) and Desorbent(D) and exit ports for Raffinate (R) and Extract (E) are placed atspecific points in the series of separation beds. At defined intervals,the position of the beds is switched in the opposite direction from theflow, producing a countercurrent movement of the solid phase bedsrelative to the fluid streams. Feedstock (F) introduced into the firstbed begins to separate into components contained therein as flow ensues,with less retained species migrating in the direction of fluid flow andbeing collected at the Raffinate port. The more retained species remainpreferentially associated with the solid phase and are collected at theExtract port. By regulating the switch times and flow rates of F, D, R,and E, a standing wave pattern is established, allowing for continuousflow of purified products from the system.

The basic SMB process is further illustrated schematically in FIG. 1. Atthe top of the diagram, feed depicted as a mixture of solid and opencircles is introduced between chromatographic columns 1 and 8 with thedirection of fluid flow clockwise, and column switchingcounterclockwise. In Zones II and III (columns 7, 8, 1, and 2)separation of species occurs, and the open circles exit the system inthe raffinate at the three o'clock position. Desorbent is introduced atsix o'clock, and flows clockwise into the system. As the columns rotate,purified closed circle species is drawn off in the extract at the nineo'clock position.

Historically, SMB has been typically applied to large scale industrialbinary separations. The process in the '589 patent was applied to theindustrial scale separation of cyclohexane from n-hexane. More recentlySMB has found favor in separating sugar isomers, hydrocarbons, solvents,and other industrial applications. Many of these industrial devices,like the original '589 device, employ variations of mechanical rotaryvalves in effecting column switching. The valve components are arrangedso that at any given valve position, multiple inlet and outlet flows aredirected to predetermined columns, and advanced one positioncorrespondingly with each rotational step. Such rotary valves aredisclosed in U.S. Pat. Nos. 6,719,001, 4,574,840, and 4,614,205. Toemphasize the intended scale of some of these devices, refer to U.S.Pat. No. 3,040,777 which describes a valve occupying an area of 64 sq.feet and weighing 10 tons.

One of the frequent problems with rotary valves is a tendency to leak atthe junction of the rotating member and the stator element. To eliminatethis problem many attempts have sought to replace a mechanical valvewith a complex series of interconnecting individual valves. Two suchvalve systems are disclosed in U.S. Pat. Nos. 4,434,051 and 5,635,072.For scale-down of SMB using networks of individual valves, anotherproblem arises, namely, accumulating too much void volume in theconnecting lines which interferes with separation. Another U.S. Pat. No.6,544,413, however, discloses a plural valve device having clusteredvalve assemblies of four valves for control of inputs/outputsproximately to each chromatographic bed. It has the advantage ofreducing volume of liquid for small scale SMB systems. U.S. Pat. No.6,979,402 discloses a device in which cross-over conduits are replacedentirely by connecting channels machined into the top and bottom platesof the rotary valve body and aligned with column ports to create an SMBfluid loop, thus reducing void volume. However, it is unclear from thisdisclosure how small the contact surface of the valve components can beto obtain adequate sealing.

There has been a recent trend in scaling SMB down to pilot and sub-pilotvolumes, as more sophisticated applications have arisen in the finechemicals and pharmaceutical fields requiring milligram-to-gram levelquantities of product. Several recent applications of SMB to thepurification of pharmaceutically active diastereomers and enantiomershave been disclosed in U.S. Pat. Nos. 6,462,221, 6,461,858, 6,458,995,and 6,455,736. Uses of new chiral resins in SMB for binary separationsof such molecules are becoming commonplace. SMB is also beginning to beconsidered for purification of biomolecules from complex mixtures. Forexample, purification of monoclonal antibodies using SMB has beenreported in Gottschlich, et al., J. Chromo. A, 765 (1997) 201 anddisclosed in WO 2004/024284. Standing wave strategies based on SMB havebeen developed for insulin purification, as reported by Mun, et al.,Biotechnol. Prog., 18 (2000) 1332.

The Protein Structure Initiative is a national effort to determine thethree-dimensional structure of a wide variety of proteins. Thisinformation will accelerate the discovery of protein function and enablefaster development of new therapies for treating genetic and infectiousdiseases. Begun in 2000, this decade-long project has recently enteredits second phase. In the second phase, ten new centers will participatein addition to the first nine. One of the significant challenges is todevelop methods of purifying target proteins from complex cell extractsin small (10-100 mg) quantities, in high purity (greater than 90%).Basic structural analyses by techniques such as x-ray crystallographyand NMR spectroscopy require that standard. A clear need exists for anSMB device that is capable of purifying target small molecules andbiomolecules in low (multi-milligram) amounts which avoid mechanicalcomponents that leak and large void volumes that interfere with purity,while embodying the hallmarks of SMB, which are controlled purity andcontinuous production.

SUMMARY OF THE INVENTION

The present invention fulfills design requirements for economy in liquidtransfer from column to column and for simplicity in valve functionwithout a need for moving parts prone to leaks. It is an object of theinvention to provide such a device scalable to accommodate small columnscapable of purifying milligram-to-gram quantities of target substances.It is a further object to provide an SMB device having simple, easilyprogrammable controls, and which is easy to repair and maintain.

The basic element of the invention is the valve system. In its simplestform, a group of valves or valve block consists of two plates havingspecialized grooves and bores wherein a pliant diaphragm is used tocontrol fluid flow. A metal or plastic lower plate contains a commongroove etched into a flat upper surface terminating at each end invertical bores extending perpendicularly to the lower surfaceterminating in fluid connectors. The lower plate also has one or morebores offset from the common groove. A valve diaphragm made of apressure responsive material is placed on top of the plate. An upperplate contains bores from its top surface extending through the plateand terminating in small recesses or dimples on the undersurface. Theserecesses are in alignment with the offset bores and also encompass aportion of the common groove on the lower plate. The components arefastened together. When pneumatic pressure is applied to the upper platebores, the diaphragm is urged against the upper surface of the lowerplate effectively sealing the bore.

In operation, inlet liquid flowing into one of the lower plate borestravels up, and traverses the common groove before exiting the secondlower plate bore. If liquid is to be withdrawn from or introduced intothe system through an offset bore, the pneumatic pressure is releasedand the diaphragm deflects partially into the recess, exposing a liquidcommunicating channel between the offset bore and the common channel;causing liquid to exit or enter through the corresponding offset bore.

The simple valve configuration described above enables control ofmultiple fluid ingress and egress streams between two adjacentcomponents, such as chromatography columns. In SMB it is necessary toswitch a minimum of four flow streams (Feed, Raffinate, Desorbent,Extract) between a minimum of four columns. Thus, in order to constructthe connections required for SMB with the above simple valveconfiguration, separate ingress and egress lines would be required foreach pair of columns, which would in turn require separate flow controldevices, manifolds, or additional valves for each line and addsignificant void volume. The invention herein described overcomes theselimitations by incorporating a series of ingress and egress channelsinto the valve block to enable access to any number of columns throughcommon lines. This valve configuration of the present invention hasseveral advantages in miniaturized SMB. First, all of the column inputsand outputs for any column can be arranged along a common groove joiningthe flow between any two columns. Second, inlet and outlet valves can bearranged in high density thereby minimizing void volume in the system.Third, the device has no moving parts per se. Maintenance is virtuallynon-existent. In the event that the diaphragm becomes scratched andbegins to leak, it is easily replaced.

In the annular configuration of the present invention, a number ofingress and egress channels are arranged substantially in concentriccircles to minimize the path length of the connecting flow pathwaybetween columns. In standard SMB, defined as a system having acontinuous uninterrupted liquid loop interconnecting all of the columns,there are minimally four columns, but systems can be constructed to anynumber. Some known SMB systems operate with as many as thirty twocolumns. In what is here termed affinity or discontinuous SMB mode,there are two or more separate discontinuous liquid loops operatingsimultaneously. In either case, the complete system has two annularvalve block modules, one in inverted orientation with respect to theother, and aligned so that columns are sandwiched between the twomodules.

The annular valve block module containing a plurality of valves is madeup of several functional layers which are securely fastened together.The upper plate has top and bottom surfaces and containing a number ofbores arranged radially and extending perpendicularly from the topsurface to terminate in recesses on the bottom surface. The bottomsurface interfaces with a pliant diaphragm, which in turn contacts theupper surface of a fluid transfer plate. The fluid transfer plate hasupper and lower surfaces with grooves etched on both surfaces. Thegrooves on the upper surface, the functional equivalent of the commonchannel referred to above, have bores within the groove at the very endsof the groove, so that each groove terminates in a bore at both ends.The upper surface grooves are preferably facing to the perimeter of thefluid transfer plate to allow maximum space for the columns in thecomplete SMB unit. There are also a series of offset bores arrangedspacedly in liquid flow proximity to the grooves, and are alignedsubstantially centrally with the recesses of the manifold. These boresextend through the fluid transfer plate and communicate with two or moreconcentric circular ingress and egress channels etched on the lowersurface of the fluid transfer plate. The concentric channels may becontinuous or consist of circular arcs that terminate at or near theoffset bores that communicate with adjacent columns.

The valve diaphragm is comprised of a pliant pressure responsivematerial, preferably a fluorpolymer, disposed between the upper platebottom surface and fluid transfer plate upper surface. When pneumaticpressure is applied, this diaphragm is urged against the fluid transferplate in sealing engagement, and when pressure is released, it deflectsinto the recess in the upper plate due to pressure from the fluid flow.

An anchor plate which forms the bottom layer in the assembled moduleprovides strength to ensure sealing of the interfaces between layers ofthe module. It has upper and lower surfaces containing columncommunicating bores in corresponding alignment to the vertical fluidtransfer plate bores, and access ports that align with the ingress andegress channels on the fluid transfer plate. Optionally, there may be abarrier membrane forming a sealing interface at the lower surface of thefluid transfer plate which forms a barrier wall for the circularchannels also containing column access ports to communicate with alignedcolumns and the fluid ingress and egress channels.

In affinity or discontinuous chromatography the process requirements aredifferent than for standard SMB. For improved purity and yields, it isimportant to thoroughly wash columns to which target is bound beforeeluting. Further it is essential to wash columns from which target hasbeen eluted to regenerate the columns for the next cycle of adsorption.This is accomplished by including one or more isolated loops which allowwashing of columns when they enter into the pre-elution and regenerationzones. The annular valve block module for discontinuous SMB hasstructures for upper plate, valve diaphragm, optional barrier plate, andanchor plate substantially identical to that for standard SMB.

The fluid transfer plate has different features. In one embodiment thegrooves on the upper surface are radial, U-shaped, and terminate on oneend in a first column communicating bore extending to the lower surfaceof the fluid transfer plate, and at the other end, a second offsetcolumn communicating bore in substantially central alignment to arecessed bore of the pressure manifold in liquid flow proximity to theU-shaped groove. In a preferred embodiment there are a total of eightsuch bores for each column, four of which reside on one valve blockmodule for the inlet streams, and four of which reside on the othervalveblock module for the outlet streams. As a flow stream enters thevalve block it has access to any of the columns via its ingress channel,which communicates with the offset bores in the fluid transfer plate.When it encounters an open port it proceeds through the offset bore tothe upper surface of the fluid transfer plate, into the U-shaped groove,and then to the column inlet port. After exiting the column, it flowsthrough the U-shaped groove on the other valve block module, through thebore of any open outlet valve, through the appropriate egress channel,and out of the valve block to the collection reservoir.

In addition to the inlet and outlet valves comprised of offset borescommunicating with the U-shaped groove, another valve is required forcontrolling the flow of one column to the next. This valve, the cutoffvalve, is constructed by interrupting the U-shaped groove on the uppersurface of the fluid transfer plate, such that a gap is formed,preferably at the downstream end of the last outlet valve and before thefirst inlet valve of the next column. The valve is actuated via acorresponding dimple in the lower surface of the upper pneumatic platewhich spans the ends of the interrupted channel and does not utilize anoffset bore. Thus when pneumatic pressure is applied the diaphragm sealsthe gap between the ends of the channel and flow to the next column isprevented. The cutoff valves enable one or more columns to be segregatedfrom fluid contact with the rest of the columns for the purpose of usingdifferent solvent or buffer streams, such as in discontinuous protocolsrequiring different buffer conditions for binding, washing, elution, andregeneration of column beds.

A complete standard SMB device for recovery of components of feedstockseparated in a raffinate or extract has means for providing a pressureforce to the pressure manifold, and fluid diversion means to regulatepressure against the various individual valve sites on a valve diaphragmcorresponding to the recesses of the pressure manifold. The device alsoutilizes two annular block modules wherein one module is aligned with asecond module in inverted orientation. The modules are interconnected toa plurality of chromatographic columns attached to column communicatingbores in equal number, so as to create an end-to-end continuous fluidloop in the system. One or more check valves are positioned to directfluid flow in the desired direction. Pressure means such as an airdriven cylinder or a pump propels liquid from reservoirs into the accessports at pre-selected positions. Outlet restrictive means permitregulation of the internal operating pressures, and also permitadjustment of the relative output volumes. Outlet restrictive meansincludes a pump as well as devices which constrict the diameter of theoutlet ports. The device is held together by fastening means to traversethe block modules to maintain alignment and prevent leaks in fluidmovement. Finally, there are control means to switch the valvingaccording to a pre-determined sequence.

The device for purifying biological substances by affinity ordiscontinuous chromatography similarly has means for providing apressure to a manifold having fluid diversion means to regulate pressureagainst the individual valve sites on a valve diaphragm corresponding tothe recesses of the pressure manifold for discontinuous mode. The deviceis composed of two annular block modules wherein one module is alignedwith a second module in inverted orientation interconnected by aplurality of chromatographic columns attached to the columncommunicating bores of equal number. Pressure means are provided todirect flow of liquid to the pre-selected access ports of the modules,and egress means permit collection of wash solution from theregeneration zone and eluate and raffinate. Fastening means traversingthe block modules to maintain alignment and prevent leaks in fluidmovement. One or more check valves may optionally be placed in line todirect and enhance proper flow. Finally, control means direct theswitching of valving according to a predetermined sequence. In bothstandard SMB and discontinuous modes, the means of exerting pressure inthe pressure manifold may be either pneumatic or hydraulic.

The same device may also be configured to perform various combinationsof standard SMB and discontinuous modes. For example, a discontinuousloop may be included in a two-column a section of an eight-column SMBdevice for the purpose of cleaning the columns, with the other sixemployed in a continuous SMB separation. As another example, adiscontinuous protocol may be employed as the first step inpurification, followed by standard SMB using a different protocol in thesame device as the second step.

In one embodiment, there is an advantage in providing a separatesolenoid valve block and manifold for each valve block module. The typeand pressure rating of the solenoids can easily be modified bysubstituting a different block in different applications. This has, ofcourse, the disadvantage of requiring individual pneumatic or hydraulictubes to be extended from the solenoid block to each pneumatic valveport on the device. Also individual wires to power each solenoid may berequired.

In a preferred embodiment, these tubes and wires are eliminatedcompletely so that a compact device contains within the valve moduleitself all pneumatic and electronic valve control features of thesystem. Such device has several component members, a pneumatic manifoldplate assembly consisting of a pneumatic plate, an electronic circuitboard, and a solenoid valve; a fluid transfer plate for directing flowof liquid through the columns and adding or drawing off liquids from thechromatography stream; a valve diaphragm for closing or opening selectedvalves at various intervals; a barrier plate to seal fluid ingress andegress channels; and an anchor plate to provide fluid attachment meansfor egress and ingress of fluids from the system.

The pneumatic plate has a first member having a top surface, bottomsurface, and a core. The top surface has a plurality of raised,elongated pedestals arranged in parallel. The upper surface of thepedestals contains a plurality of bores arranged rectilinearly andextending perpendicularly from a pneumatic valve port terminating inrecesses on the pneumatic plate bottom surface. A second perpendicularbore extends from a pneumatic manifold port on the upper pedestal tocommunicate with a transverse pressure channel embedded in the core ofthe plate. A pneumatic exhaust channel is contiguous to a pedestalrelieves pressure to selected valves. The second member of the pneumaticplate is an electronic circuit board which has slots the size and shapeof the pedestals so that when placed on a receiving surface on top ofthe first member pedestals, exposes the pedestal bores in alignment ofthe two members, which are secured by fastening means. The circuit boardhas receptacles for a third member solenoid body electrical conduitswhich mate together to form an electrical connection. The pneumaticvalve ports, manifold ports and exhaust channels are adapted to alignwith the corresponding ports on the solenoid valve, and the contact ofport orifices are sealed by gaskets. The precise positioning of ports onthe upper surface of the pedestals will be dictated by the position ofcorresponding solenoid orifices for the type and brand of solenoidselected.

A second component of the valve module is a fluid transfer plate havingan upper surface and a lower surface, the upper surface displaying aplurality of fluid transfer grooved channels etched thereon, terminatingat each end in substantially vertical column port bores. These ports arearranged into rows of equal number along opposite sides of the plateextending to the lower surface of the plate in alignment withchromatographic columns situated at corresponding intervals under themodule. In addition, there is an array of offset bores in liquid flowproximity to the grooved channels aligned to the recesses on the lowersurface of the pneumatic plate, and extending through the transfer plateto communicate with two or more fluid ingress and egress channels etchedthe lower surface of the fluid transfer plate.

There is a valve diaphragm composed of a pliant pressure responsivematerial disposed between the lower surface of the pneumatic plate andthe upper surface of the fluid transfer plate. The diaphragm lacks boresexcept where required for screws or other fasteners for holding theassembly together. There is a barrier plate forming a sealing interfaceat the lower surface of the fluid transfer plate, forming a lowerbarrier wall to the fluid egress and ingress channels. This plate alsohas column access bores to communicate with the aligned chromatographiccolumns and the ingress and egress channels. Finally there is an anchorplate having an upper and a lower surface containing columncommunicating bores in alignment with the chromatographic columns andthe ingress and egress channels.

The chromatography device comprises two modules oriented in invertedconfiguration with columns connected between them by fitting means andattached at bores in the anchor plate. The upper surface of the fluidtransfer plate design is different for the two modules, in order toallow the columns to be attached between them in a parallel orientation.In one fluid transfer plate the grooved channels on the upper surfaceare adapted to enable the outlet ports and cutoff valve from one columnto communicate with the inlet ports of the next column in a differentrow level. In a preferred embodiment each grooved channel thatrepresents the outlet ports for the columns on one valve block module isextended to the column outlet port one row below. This configurationenables the fluid path to proceed upward through each successive row ofgrooved channels into columns extending perpendicular to the valve blockmodules and parallel with each other. The grooved channel extension forthe topmost column outlet is different in that it communicates back tothe lowest row to complete the fluid loop. In the other fluid transferplate the upper surface grooved channels do not contain extensions, andeach column outlet port is at the same level as its row of valveoutlets, which in turn communicate to the inlet valves and inlet portfor the next column along the same level. The upper surface groovedchannels of either fluid transfer plate may interrupted by gaps havingends that can be spanned by corresponding dimples in the lower surfaceof the pneumatic plate to form cutoff valves. The ends of theinterrupted channels may be curved to provide liquid flow proximitybetween the ends when pressure is released in a pneumatic valve situatedimmediately above the gap.

The simulated moving bed device comprising the two valve modules alsohas means to apply a pressure force to a pneumatic plate having airdiversion means or manifold to regulate pressure against individualsites on the valve diaphragm corresponding to the recesses of thepneumatic plate. The manifold is a transverse pressure channel containedin the core of the pneumatic plate. The two modules, left and rightoriented, are interconnected by a plurality of chromatographic columnsattached to the communicating access bores. The device further haspumping means to direct ingress or egress flow of liquid to and frompre-selected access ports of the module, and fastening means to maintainalignment and prevent fluid or pneumatic leaks.

Control of a system in accordance with the present invention preferablyincludes a personal computer running control software which provides auser-friendly interface for a user to control the valve stateconfiguration. Preferably the control software allows the user to eithermanually control the valve configuration within a chromatography moduleor to set up and run automated scripts that direct changes in valvestates that allow the chromatography module to separate and purifycompounds. The personal computer interfaces with control electronicsthat act on control signals generated by the control software inresponse to the user inputs. The control electronics operate to actuatesolenoids that control the gas or fluid pressure applied to eachmembrane valve to control the valve states in the system. Alsocontrolled by the personal computer are pumps that direct the flow offluid through the system. The pumps receive signals relayed to themthrough an adaptor board from the computer. Together, this controlsystem configuration allows for complete automation of a chromatographicseparation process using an SMB system in accordance with the presentinvention.

In the method of purifying biological substances by discontinuous oraffinity SMB in which four zones of chromatography steps, namely,adsorption, wash, elution, and regeneration, wherein there is adiscontinuous downstream propelled flow of fluid, and a regularcountercurrent cycling of valve positions in recurrent stepwiseconfiguration, a feedstock is applied to a first column which contains atarget substance of interest. The target substance is permitted to bindto an immobilized ligand in the column through complementary affinity ofa chemical moiety appended to the solid phase. In a preferred embodimentbinding is performed in an isolated zone of one or more columns whereinthe feedstock is applied at an inlet port of the first column andunbound material is removed at an outlet port of the last column. Theisolated zone is established by closing appropriate cutoff valves toprevent flow from the column upstream of the zone and to the columndownstream of the zone. Cutoff valves between columns within the zoneare opened. Next, a majority portion of the unbound contaminants isremoved in an isolated zone of one or more columns wherein a wash bufferis applied at an inlet port of the first column and the wash effluent isremoved at an outlet port of the last column. In an alternativeembodiment the cutoff valve between the wash zone and the binding zoneis opened to allow the flow stream of wash buffer to combine with thefeedstock. The next step is eluting the target substance in an isolatedzone of one or more columns wherein an elution buffer is applied at aninlet port of the first column and the eluate is collected at an outletport of the last column. The final step is regenerating the columns inan isolated zone of one or more columns wherein a regeneration buffer isapplied at an inlet port of the first column and the effluent is removedat an outlet port of the last column. This process is repeated at eachvalve switch position of the device. It will be apparent to thoseskilled in the art that by appropriate configuration of valve positionsin the sequence by control means, the affinity mode SMB device can beprogrammed to perform standard SMB as well.

In a preferred affinity chromatography method, standard SMB havinginlets for feedstock and eluent and outlets for extract and raffinate isemployed to isocratically purify an affinity tagged protein from acomplex mixture of proteins in a feedstock by selecting a concentrationof an affinity neutralizing agent sufficient to prevent static bindingof the target to a resin contained in the multiple columns. At thisselected concentration there is still some affinity interaction betweenthe target protein and the resin sufficient to retard passage of thetarget protein through the resin in a mobile phase. This is in contrastto other proteins in the mixture not having such affinity, which readilypass into the raffinate. Thus, protein contaminants which wouldotherwise bind weakly to the resin in a static binding mode, areeliminated from the product stream. The concentration of affinityneutralizing agent is adjusted to the selected concentration in both thefeedstock and eluent, so that the entire SMB process is carried out atone isocratic concentration. The feedstock is continuously fed into theSMB device to obtain purified target protein from the extract outlet.This method is useful also for purifying proteins having endogenousselective affinity for a ligand which is bound to a column resin.Examples include molecules having immune specificity with bindingdomains for protein A and G, enzyme-substrate binding sites, orco-factor binding sites. The method is particularly efficacious forrecovery of monoclonal antibodies from cell culture, ascites fluid, orrecombinant origins.

It will be apparent to those skilled in the art that the discontinuousand isocratic SMB modes can be applied in a variety of configurationswith virtually any liquid chromatographic separation system, including,but not limited to; metal affinity, immunoaffinity, substrate affinity,anion or cation exchange, hydrophobic interaction, thiophilic, andrecombinant protein affinity tag systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating an eight column standard SMB systemin a two species separation at one point in time for a 2-2-2-2configuration.

FIG. 2 is a diagram of an eight column standard SMB system showing thevarious chromatographic zones and the position of inputs and outputs fora 3-1-3-1 configuration.

FIG. 3 is a cross-sectional view of a valve block utilizing a diaphragmunder pneumatic pressure control.

FIGS. 4A and 4B are downward and upward facing exploded perspectiveviews showing the parts and features of a basic valve block.

FIG. 5 is a perspective view of a four valve block assembly.

FIG. 6 is a downward facing exploded perspective view of a sixteencolumn annular valve module.

FIG. 7 is a upward facing exploded view of a sixteen column annularvalve module.

FIG. 8 depicts in perspective view a completely assembled sixteen columnSMB device showing the disposition of columns in relation to two valvemodules.

FIG. 9 is a cross-sectional view of a completely assembled sixteencolumn SMB device.

FIG. 10 is a diagram of an eight column SMB system showing four closedloops for preventing wash 1 solution from entering the absorption zone,desorbent (eluent) from entering the wash zone, wash 2 solution fromentering the elution zone, and feed from entering the regeneration zone.

FIG. 11 is a diagram similar to FIG. 10 for a sixteen column SMB device.

FIG. 11B is a diagram showing the flow pattern between the upper andlower modules for a sixteen column SMB device.

FIG. 12 is a plan view of the upper surface of a fluid transfer platefor use in an eight column SMB device.

FIG. 13 is a plan view of the lower surface of a fluid transfer platefor use in an SMB device.

FIG. 14 is a schematic showing various components of a control systemfor an SMB device.

FIG. 15 is a perspective view showing the relationship of upper andlower fluid transfer plates with respect to orientation of columncommunicating bores at each end of a column.

FIG. 16 is a side-by-side comparison in plan view demonstrating theasymmetry of features of the pneumatic pressure plate, fluid transferplate, and anchor plate for upper and lower modules.

FIGS. 17A-D are schematics showing the valve configuration for the firstfour cycles of standard SMB as illustrated by schematic representationof the fluid transfer plates.

FIGS. 18A and B are scans of Coomassie blue-stained SDS-polyacrylamidegels showing the protein content of various SMB fractions and standards.

FIG. 19 is an exploded perspective drawing of the right valve blockmodule of the compact device.

FIG. 20 is an exploded perspective drawing of the left valve blockmodule of the compact device.

FIG. 21 is a plan view of the upper surface of the pneumatic plate ofthe compact device.

FIG. 22 is a cross-sectional view of the upper portion of the pneumaticplate of the compact device.

FIG. 23 is a plan view of the circuit board with solenoid valves inplace and pedestal feature shown in relief.

FIG. 24 is a plan view of the upper surface of the right oriented fluidtransfer plate.

FIG. 25 is a plan view of the lower surface of the fluid transfer plate.

FIG. 26 is a plan view of the upper surface of the left oriented fluidtransfer plate.

FIG. 27 is a plan view of the lower surface of the pneumatic plate.

FIG. 28 is a left facing partially exploded cross-sectional view of theupper right corner section of the right valve block module of thecompact device.

FIG. 29 is a right facing partially exploded cross-sectional view of theupper right corner section of the right valve block module of thecompact device.

FIG. 30 is a plan view of the right oriented fluid transfer plateshowing the position of solenoids and the ingress and egress channels inrelief.

FIG. 31 is a plan view of the left oriented fluid transfer plate showingthe position of the solenoids and the ingress and egress channels inrelief.

FIG. 32 is a perspective view of the components of the carriage formounting valve modules.

FIG. 33 is a block diagram illustration of an exemplary control systemfor an SMB system in accordance with the present invention.

FIG. 34 is a block diagram illustration of a portion of the controlsystem of FIG. 33 for controlling the state of the valves in a valveblock in accordance with the present invention.

FIG. 35 is a an exemplary screen shot illustration of a graphical userinterface for defining the valve states for manual control in a controlsystem in accordance with the present invention.

FIGS. 36 and 37 are exemplary screen shot illustrations of graphicaluser interfaces for defining valve state steps for automatic processcontrol in a control system in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The principle of continuous countercurrent chromatography relies on thephenomenon of preferential retention on an immobilized sorbent substrateof one or more substances in a feedstock mixture, separation of lessretained substances, and subsequent recovery of the separatedsubstances. In standard SMB this process is repeated in a succession ofcolumns by switching zones of separation, enrichment, and regenerationin stepwise sequence. FIG. 2 is a schematic illustrating SMB in an eightcolumn 3-1-3-1 configuration. The feedstock (F) enters the systembetween Zones II and III at column 6, and after passage through threecolumns, less retained substances are collected in the raffinate (R). Ata predetermined interval, through valve switching, column 6 now occupiesthe position of column 5 in the diagram, and so on. Eluent (desorbent)which is carried by the left-to-right flow in the system through columns2 and 3 releases the more retained substance which is collected in theextract (E; also herein referred to as the eluate or product in affinityor discontinuous mode). Thus, the apparent movement of columns iscountercurrent to the direction of flow.

For large scale industrial systems, the bed volume is so great comparedto void volumes of liquid between beds that even elaborate valvingsystems involving extensive conduit do not interfere with the process.As SMB is scaled down in size, void volumes become significant relativeto bed volume, and at the level where total bed volume may be only a fewmilliliters, void volumes are crucial. The present invention addressesthe challenge of devising a valving system in which a high density ofindividual valves can be compressed into a very small area.

FIG. 3 shows such a pair of valves in cross-section. A valve blockgenerally 10 comprises an upper plate 12 having a connector 24 and avertical bore 22 terminating in a recess 20. A fluid transfer plate 16has a common groove 13 and an offset bore 30 (the offset represented bydotted lines) which communicates with an access port 32. At the bottomof the valve assembly is an anchor plate 26. Sandwiched between theupper plate 12 and the fluid transfer plate 16 is a pliant diaphragm 14,preferably composed of flexible pressure responsive material such as FEPor PFA fluoropolymer. In operation, when pneumatic or hydraulic pressureis applied the diaphragm 14 is pressed down over the vertical offsetbore 30 and common groove 13 spanned by the recess, effectively sealingthe gap between the offset bore and the common groove and preventingflow from the access port 32 below. The left valve in the diagram isshown in closed position. In this schematic the closed position of thediaphragm is meant to indicate that it is pressed against the uppersurface of the fluid transfer plate to seal the common groove and offsetbore. When the pneumatic pressure is vented, fluid pressure in thesystem causes the diaphragm 14 to deflect upwards into the recess 20forming a communicating bridge between the orifice of the offset bore 30and the common groove 13, which allows the fluid stream 28 to flow fromthe access port 32 through the offset bore into the common groove. Asimilar valve design, differing from the valve of the present inventionin that movement of the diaphragm was controlled by alternating pressureand vacuum, was used commercially in synthesizer instrumentsmanufactured in Germany during the late 1980's. Subsequently, U.S. Pat.No. 5,203,368 issued and discloses a virtually identical device.

The foregoing valve strategy can be adapted to more a complexapplication capable of chromatographic separations. FIGS. 4A and 4Billustrate a valve block having column communicating bores and fourvalve sites. FIG. 4A is an exploded view showing the spatial orientationof the parts. FIG. 4B is an exploded view from the opposite perspective.The upper plate 40 has nipples 42 fastened thereto, providing access topneumatic pressure through a bore terminating in a recess 41 (See FIG.4B) on its lower surface. A fluid transfer plate 44 is situated belowthe upper plate 40 with a diaphragm 58 sandwiched between it and thefluid transfer plate 44. There is a linear common groove 46 etched onthe upper surface of the fluid transfer plate 44, terminating invertical bores 48 passing though the fluid transfer plate 44 (as shownin FIG. 4B). In addition there are four vertical liquid transfer boresarranged along the common groove 46 in liquid communicating proximity tothe common groove 46. The term “liquid communicating proximity” hereinmeans that the distance from a fluid transfer bore to the common grooveis close enough that when pneumatic pressure is released the internalfluid pressure is sufficient to deflect the diaphragm upward into recesscavity thereby opening a liquid path between the bore and the commonchannel, but far enough apart to seal off such path when pneumaticpressure is applied. The base of the device is an anchor plate 60 havingsix bores (the two terminal bores at the ends of the common groove, andthe four liquid transfer bores in alignment with the corresponding boresof the fluid transfer plate 44. Liquid inlet/outlet connectors 56 areselected from conventional fittings. The ones represented in the figuresare compression type fittings having a stem 52 and a plunger flat headtip 54 designed to engage a seating surface within each bore to form anaccess port. FIGS. 4A and 4B also show a series of aligned peripheralbores 62, 63, 65, and 67. Bolts having threaded ends are inserted in thethreaded aligned bores to secure the layers in sealing engagement.

Minimally four valve blocks of the type described hereinabove may bearranged in a grouping as shown in FIG. 5 to construct an SMB device.Each of the valve blocks is interconnected to two others through columns70 mounted therebetween to form a continuous circular loop through thesystem. Columns are arranged head-to-tail such that the outlet of onecolumn is connected to one end of a valve block and the inlet of thenext column is connected to the other end of the valve block. Each ofthe liquid transfer ports 71 in each valve block is assigned F, R, D,and E inputs and outputs. In operation, at any given time one valve isopen in each block, and the other three are closed. At the end of apredetermined switching interval, the open valve closes and anothervalve opens in the desired sequence. Flow may be controlled by use ofconventional pumps for the inlet and outlet streams. In a preferredembodiment, check-valves 74 can be inserted at either the inlet oroutlet side of one or more columns to prevent bidirectional flow ofinput liquids. These may conveniently be placed inline of the columnconnectors 72, which constitute conventional fittings.

A more complex standard SMB device is depicted in FIGS. 6 and 7. Thisdevice is shown in a sixteen column configuration, but the number ofcolumns may be any number four or greater. Commercial SMB devices aretypically offered in column capacities from four columns to thirty two.An annular configuration is chosen to minimize void volume in thesystem. An annular valve block module, generally 100, comprises severallayers of machined components firmly compressed together to form acylindrical body. The layers are illustrated in FIGS. 6 and 7 in upwardand downward facing exploded perspective views.

The uppermost layer is an upper plate 102 having 32 or more annularlyarranged bores 104 (FIG. 6) in two or more concentric configurations,extending from the upper surface of the plate and terminating in arecess on the bottom surface 103 (FIG. 7). Press fit nipples 106attached to the bores 104 provide a coupling appendage for connecting asource of pneumatic pressure. A fluid transfer plate 110 is situatedbelow the upper plate 102. There are a series of eight U-shaped grooves112 etched onto the upper surface of the fluid transfer plate 110 andarranged annularly facing the outer perimeter of the plate. Thesegrooves terminate in column communicating vertical bores 114 at eachend, which extend from the upper surface to the lower surface (114, FIG.7). A group of four or more concentric ingress and egress channels 116(FIG. 7) are etched into the lower surface of the fluid transfer plate110. Arranged spacedly along the U-shaped grooves in liquidcommunicating proximity are four or more liquid access ports 116extending from the upper surface to the lower surface of the fluidtransfer plate, the ports being aligned to communicate individually witheach of the concentric channels.

The U-shaped grooves 112 may have square or rounded walls, and may bearranged in other geometries such as Vs or semi-circles. In theminiature SMB devices the grooves and concentric channels may be 1 mm orless in size.

A valve diaphragm 124 is disposed between the upper plate 102 and thefluid transfer plate 110, to form a sealing surface between the lowersurface of the upper plate and the upper surface of the fluid transferplate. The diaphragm is composed of a pliant pressure responsivematerial. Elastomeric Teflon such as PFA and other fluoropolymermembranes responsive to less than 20 psi of pressure differential aresuitable in this application.

An anchor plate 125 is positioned below the fluid transfer plate 110 andhas bores extending from the surface of the plate to the lower surfacethereof. The column access ports 128 are arranged in a circle andcorrespond to and are aligned with the corresponding bores 114 at theends of the U-shaped grooves 112 in the fluid transfer plate 110. Fourinlet and outlet bores 122 extending through the body of the anchorplate 125 are positioned to communicate with each of the four concentricchannels 116. The concentric ingress and egress channels are assigned toeach of F and D reservoirs, and R and E receptacles in no particularorder. Thus, there is common input and output source for all the valvesthat feed from and to each column position. Optionally, there also ventports located in each channel immediately opposite the feed bores 122,to allow escape of air from the channels during filling. Optionally,there may be a barrier membrane or plate 130 disposed between the anchorplate 125 and the fluid transfer plate 110 to provide inlet/outlet bores120 and column communicating bores 132 to the anchor plate 125. In thisconfiguration the barrier membrane or plate becomes the lower wall ofthe concentric channels 117.

The anchor plate is fitted with couplings accommodating each columncommunicating and inlet/outlet port, as described above for the valveblock assembly. FIGS. 6 and 7 show the compression type fittings 133having a stem 134. Such fittings provide a good liquid seal withoutaltering the internal diameter of port and connector. Several types andbrands of such fittings are conventionally available. The figures alsoshow the relative position of the columns 136.

Referring to FIG. 8, a full SMB fluidic system according to the presentinvention, generally designated 200, comprises two annular valve blockmodules 206 and 206′ respectively aligned in inverted orientation. Themodules are interconnected by a plurality of chromatographic columns 136attached at either end to a module by column connector means 132 mountedon the column communicating bores. The columns are attached inalternating orientation, with inlets of every other column attached toone valve block module and the inlets of the other columns attached tothe other valve block module. Also, inline of the columns are checkvalves to ensure unidirectional flow of liquid in the system. The upperplate 102, fluid transfer plate 110, and anchor plate 125 of the topmodule are shown in reverse order in the inverted bottom module as 125′,110′, and 102′. Vertical inlet/outlet conduits 202 connect each accessport (122, FIG. 7) pair of the upper and lower modules throughconnectors 132 on each end. Each vertical feed conduit 202 is providedwith a T stem 204 connected to an input reservoir or an outputreceptacle. Normally, liquid is delivered to or from the system via thevertical feed conduits under pressure using in line pumps. F, D, and Eare typically on flow rate control, i.e. pumps, It is possible to have Ron flow control as well. A suitable pump for this application includes apiston type chromatography pump, preferably using a downstreambackpressure regulator and optionally a pulse dampener to minimize flowpulsing.

FIG. 9 depicts the SMB fluidic system shown in FIG. 8 in cross sectionalview. The upper plate 102 has recesses 103 shown partially at aforeground section. The diaphragm 124 is sandwiched between the upperplate 102 and a fluid transfer plate 110. A portion of the U-shapedgroove 112 is shown in closed position, and terminates in a columncommunicating bore 114 which extends in alignment through the anchorplate 125, a column connector 244 and into the column 132. Symmetricalconfiguration is presented left and right, and invertibly for the lowermodule. Unidirectional flow ensures a continuous loop in which physicaltop to bottom flow in one column becomes bottom to top in the nextcolumn. Inputs and outputs to the loop are not shown because thesestructures are not in the cross sectional plane. The flow patternbetween modules is illustrated in FIG. 8B for a sixteen column device.The dotted lines indicate the concentric channels located on the lowersurface of the fluid transfer plate. A valve is positioned at each pointwhere the dotted lines intersect a flow on the U-shaped groove.

The upper plate and anchor plate may be machined from aluminum,stainless steel, or a variety of plastics. Aluminum alloy 6061-T6 isparticularly suitable for this application. Contact surfaces, namely,the lower surface of the upper plate, the upper and lower surfaces ofthe fluid transfer plate, and the upper surface of the anchor plateshould preferably be polished to a #4 microinch RMS surface roughness,in order to seal properly. The fluid transfer plates may be machinedfrom a selection of hard plastics. PEEK or PCTFE are preferred.Diaphragms and membranes can be fabricated from 5-10 mil sheets offluorocarbon polymers, polypropylene. Fittings are all conventionallyavailable in PEEK, PTFE, ETFE, PCTFE, and other plastics. All of theabove materials are resistant to corrosives and solvents, durable, andrigid, except for the pliant, pressure responsive membranes anddiaphragms. The anchor plate can be manufactured for thickness andphysical strength and serves as a rigid base plate allowing the otherlayers to be made as thin as possible to reduce void volume everfurther.

An important aspect of the present invention is adaptation of theprinciples of affinity chromatography to SMB. In standardchromatography, the basis of separation is differential elution ofspecies as a function of some generalized physical or chemical property.For example, in size exclusion chromatography separation is on the basisof molecular size and shape. In ion exchange chromatography separationis based on charge. In standard chromatography based on general chemicalproperties, separation of the desired species from complex feed mixturesis not highly discriminating. All proteins of a particular molecularsize or charge will elute under similar conditions, thus reducingpurity. This explains why high purification to greater than 90%homogeneity often involves multiple purification steps customized foreach target protein.

In affinity chromatography, the target molecule contains a chemicalmoiety which specifically interacts with a second moiety immobilized ona column substrate. Recombinant proteins can be engineered to containspecific peptide or other chemical moiety which act as affinity tagsthat bind specifically to immobilized ligands. If the affinity tag issomething either not naturally occurring or not likely to be present onproteins in the cellular contents in which the target protein isexpressed, a high level of purity can be obtained using a relativelystandardized protocol. This has significant implications in structuralproteomics, where the challenge is to purify large numbers of proteinsat multi-milligram scale for x-ray crystallographic or NMR spectroscopicanalysis.

A number of such affinity chromatography systems have been describedwhich can be readily adapted to an SMB purification stratagem. U.S. Pat.Nos. 5,310,663, 5,284,933, and 4,569,794 disclose a hexahistidyl taghaving specific affinity for a resin incorporating a metal chelate. Afusion protein of interest having the six histidine tag preferentiallybinds to a Ni⁺⁺ chelate on the solid phase. The binding affinity forenzyme and substrate is exploited when a fusion protein ofglutathione-S-transferase recognizes and selectively binds to aglutathione-conjugated resin, as described in U.S. Pat. No. 5,654,176and Smith, et. al, Gene 67: 31(1988). Other examples of affinity systemsinclude a peptide fusion system [Raines, et. al, Methods Enzymol., 326:362 (2000)], cellulose binding domain fusion proteins (U.S. Pat. Nos.5,719,044 and 5,202,247), mutant streptavidin binding peptide (U.S. Pat.No. 5,506,121), and calmodulin binding protein fragment [FEBS Lett. 302:274 (1992)]. The advantages of SMB in these affinity systems are threefold: (1) the ability of SMB of sufficient column number to fine tunecollection of a high purity target fraction, (2) the inherent capabilityto process cell extracts continuously, and (3) the capability of SMB tomore efficiently utilize the solid phase. These advantages areespecially significant when purifying target from cell extracts ofinsect cultures where expression levels often are below 1% of totalcellular protein.

The requirements for affinity SMB are different operationally because ofthe necessity of isolating portions of the otherwise continuous loop instandard SMB, if the system is to mimic standard batch type affinitychromatography. FIG. 10 is a schematic of one embodiment of affinity SMBfor an eight column system. Four chromatographic zones are depicted,regeneration, elution, wash, and adsorption. The regeneration zonecomprises the column 1 and 2 positions, elution takes place in thecolumn 3 and 4 positions, the wash occurs in the column 5 and 6positions, and adsorption occurs in the column 7 and 8 positions. Theterm “column positions” rather than columns per se is used because theactual columns participating in each functional step keeps rotating asfor standard SMB. Thus, after a predetermined interval, column 7occupies the functional position of column 6 as depicted, and so on.Like standard fluid SMB, flow, as indicated by the arrows, is generallyleft-to-right in contrast to the columns which appear to be moving in aright-to-left direction. The size of the system can encompass fewer orgreater numbers of columns, depending on how many columns are assignedto each chromatographic zone. For example, FIG. 11 is a schematicdrawing showing sixteen columns, and assigning four columns for theelution zone, four columns for the wash zone, six columns for theadsorption zone, and two columns for the regeneration zone. Withsufficient column number encompassing several within a given zone, morethan one wash/desorbent can be introduced into and exit a flow isolatedzone.

In operation, according to FIG. 10, feedstock containing the species tobe separated is loaded onto column 7, where it is statically adsorbed tothe affinity matrix of the column. Adsorption continues into column 8.Unbound species are largely eliminated from the system in the raffinateexit port at column 8. Column 7 then “rotates” by valve switching intocolumn 6 position and then the column 5 position. In this zone,extensive washing of the column takes place. Rightward directed flowcarries residual unbound or weakly bound contaminants through the systemand they are eliminated as Wash 1 Effluent after exiting column 6. Sincethis zone is a closed loop the flow rate of the Wash 1 stream can beadjusted independently of the other streams and therefore a greatervolume of fluid can be flushed through the columns in a given period formore thorough removal of contaminants. In the next two valve switchings,original column 7 enters the original column 3 and 4 positions. Eluentis introduced at column 3 and exits at column 4. In affinitychromatography, even small residuals of desorbent may interfere withefficient binding at columns 7 and 8. Therefore, the eluent is also aclosed fluid loop, and the desorbent is not permitted to enter theabsorption zone of the system. After elution of the desired target, theoriginal column 7 passes into a second wash zone for regeneration of thecolumn which removes desorbent, and prepares the column for the nextround of adsorption. This is also a closed fluid loop to eliminatedesorbent from the system. Thus, there are four independently controlledloops in this configuration, each forming a zone having conditions toenable efficient adsorption, washing, elution of the purified target,and regeneration of the solid phase. For purposes herein the terms“eluent” and “desorbent” are deemed identical and refer to the samething; differentiated only because they are expressed in the literatureas different terms which have the same meaning.

The apparatus of the present invention for performing affinity SMB isvery similar to that for standard SMB, but with two significantdifferences. Four new input/output streams are incorporated by addingcorresponding valve ports and ingress/egress channels. These streamsenable the configuration of the wash zones in which different bufferscan be introduced and removed for the wash and regeneration steps. Acutoff valve is also required to prevent flow between columns for theestablishment of isolated zones (closed loops). There are thus ninetotal valves present on each U-shaped groove, rather than the minimum offour for a standard SMB system. The additional flow streams and cutoffvalves also enable additional configurations of standard SMB, such asthe incorporation of internal recycling flow control, thus increasingutility for this mode as well.

FIG. 12 is a plan view of the upper surface of a fluid transfer plate110 for affinity or standard SMB. There are a series of four U-shapedgrooves 112 arranged annularly facing the outer perimeter of the plate.Each of the grooves terminates in two column communicating bores 114,which extend from the upper surface of fluid transfer plate 110 to thelower surface thereof. Each groove also contains a gap, or interruption,for the cutoff valve 115. The much preferred position of the gap 115 isdownstream of the outlet valves for the preceding column. Arrangedspacedly along the U-shaped grooves in liquid communicating proximityare eight liquid access ports 116 extending from the upper surface tothe lower surface of the fluid transfer plate 110. The dotted linecircles shown on the figure indicate the position of the columns in theassembled valve block module. As seen in FIG. 13, the lower surfacebears eight concentric channels each intersecting four of the liquidaccess bores 116 thereabove. Referring to FIG. 15, two facing fluidtransfer plates are placed in alignment and columns are positionedtherebetween in partially exploded view. Note that in the lower fluidtransfer plate the orientation is reversed so that liquid passingthrough a column communicating bore on the upper plate on the inlet legof the U-shaped groove is always paired with a column communicating boreon the lower plate on the outlet leg of the U-shaped groove adjacent toits cutoff valve, This has certain manufacturing implications, becauseit is apparent that upper and lower plates are not identical, and infact, are mirror images. Similarly, referring to FIG. 16, the upperplates of the upper and lower modules are also mirror images, as are theupper and lower anchor plates.

Valves under pneumatic or hydraulic control operate under pressureemanating from conventional manifolds. The source of pneumatic pressuremay be a common inert gas pressurized tank containing either nitrogen orhelium. In the case of affinity SMB, the complexity of control is suchthat only an individual solenoid valve controlling pressure from amanifold to a sole SMB diaphragm valve is practical. In standard SMBhaving one continuous liquid loop through the entire device, the patternof open and closed valves is exactly the same for each column position.Thus, this pattern for F, R, D, and E can be programmed for each of theeight columns, and the pressures for each eight column configuration canbe under a common pressure solenoid. The minimum number of solenoidvalve required for an eight column standard SMB system is eight ratherthan the 32 required if each diaphragm value is under separate solenoidcontrol. Solenoid valves suitable for SMB applications are typicallythree way (vent to atmosphere in position 3) normally open valves. Onecommercial source of such valves is Spartan Scientific, series 1590.

In another embodiment of the present invention, a compact valve moduledesign eliminates all external control wires for solenoids, and theexternal pneumatic lines controlling the state of the pneumatic valves.This is a significant improvement in design for situations as in aresearch laboratory where space is limited and instrumentation istypically compact. The small size of the present instrument lends itselfwell to research applications in chromatography where milligram amountsof the desired molecular product can be purified on small columns havinga bed capacity of one or a few milliliters. The module components arefunctionally equivalent to the annular embodiment, and operate on thesame principles, but are configured differently. The parts may befabricated of the same materials and utilize the same sources ofpneumatic force and pumping means.

FIGS. 19 and 20 are exploded perspective views of the compact unit rightand left module, respectively, generally 400, illustrating the sixfunctional plates of the modules utilized in the fully assembled SMBunit, plus an optional cover plate 402. Note that all the plates arerectangular rather than annular in shape, for reasons that will beevident hereafter. The outermost two plates are the two components of afirst member of the valve module, and consist of an electronic circuitboard 404 and a pneumatic manifold plate 406. The top surface of thepneumatic manifold plate 406 has a plurality (in this case four) raisedelongated pedestals 412 and a plurality of posts 418 adapted so that thecircuit board 404 will lie flush to the top of the pedestals 412 whenassembled. The posts 418 have threaded bores in the top surface forsecurely fastening the circuit board 404 to the posts 418. The circuitboard 404 has slots 408 the same size and shape of underlying pedestals412 thereby exposing the top surface of the pedestals 412.

Referring to FIG. 21, a plan view of the top surface of the pneumaticplate 406 shows further detail. Each solenoid valve port must beconnected to the pedestal 412 and also a two-lead electrical connectionmust be made to the circuit board 404 to be fully functional. Thesolenoid valves used in this embodiment are of the type having arectangular valve body, are normally open, and have a gasketed port forapplying pneumatic pressure to pneumatic valves in the device to controlpatterns of fluidic flow, a gasketed port for incoming gas from apressurized gas source, and a vent. FIG. 21 shows a series of bores inthe top of the pedestals 412, two of them in a horizontal plane 422, andtwo in a vertical plane 424. The two horizontal bores 422 are threadedand permit securing the solenoid valve body to the pedestals 412 byscrews. The two vertical bores 424 and 425 form a sealing engagement ofthe gasketed ports for delivery of air pressure and an incoming sourcepneumatic pressure respectively. A vent for exhausting pressure isprovided by an exhaust channel 426 running the length of the pedestals412, open to the atmosphere. In a cross-sectional view, FIG. 22 showsthe pedestals 412, the pneumatic bores 424, a manifold channel 425, andthe exhaust channel 426. This figure also illustrates the circuit boardmounting posts 418 and the threaded bore 419 for mounting the circuitboard to the pneumatic plate 406.

Referring again to FIGS. 19 and 20, the circuit board 404 has a seriesof bores 428 adjacent to the top and bottom edges of the circuit board,and adjacent to each of the slots 408. These bores contain receptaclesinto which prong power leads insert to connect the solenoids to thecircuit board. Each solenoid requires two such prongs to closeelectrical contact. FIG. 23 illustrates the assembly of solenoids 405onto the circuit board 404. Dotted lines represent the outline of thepedestals. The distance between pneumatic valve ports is essentially thewidth of the solenoid valve body, so that the array of solenoids in itsmost space saving configuration is rectilinear.

A pliant diaphragm 430 is disposed between the pneumatic plate 406 and afluid transfer plate 432, as shown in FIGS. 19 and 20. This membrane isa fluorocarbon film identical to that used in the other embodiments ofthe device, and serves the same purpose. The diaphragm is a pliantpressure responsive polymer, sufficiently pliant to permit be deflectedwhen pneumatic pressure is relieved at a valve site, permitting flowfrom or into valve ports. In closed position, pneumatic pressure againstthe diaphragm exceeds the fluid present in fluid ports and preventsflow.

FIG. 19 shows the position of the right module fluid transfer plate 432disposed between the diaphragm 430 and a sealing plate 434 in explodedview. The sealing plate 434 is optional if bores are countersunk in theanchor plate to ensure that fittings do not have to seal directlyagainst the ingress and egress channels 452 directly. A plan view of thefluid channels etched into the top side of the right module fluidtransfer plate 432 is shown in FIG. 24. Fluid transfer channels aredepicted by four separate double line structures, ignoring the cut offgap zones 438. Each channel terminates at its ends in a column accessport 436. Note the curved portion 440 of each channel, termed thecross-over channel which directs flow from a column access port in therow on one side of the fluid transfer plate to the row of valves abovethe row where the column access port entered the plate (in three cases)or from the column access port at the top left of the plate 436 to thebottom row of valves (in the fourth case). The serpentine channel thatconnects the access port 436 at the top left of the lower array to theaccess port at the top right of the upper array ensures that there is acomplete fluid loop among all the columns, so that the cycle can berepeated indefinitely. The small bores 444 and 446 are fluid egress andingress ports respectively for drawing off or introducing liquids intothe stream. The reverse side of the fluid transfer plate 432 is shown inexploded view (FIG. 20) and in greater detail in the plan view of FIG.25. Referring to FIG. 25, there is a plurality of ingress and egresschannels 452 etched into the lower surface of the fluid transfer plate432. Each channel is shown with four bores 444 which correspond on thebottom surface to the same bores depicted on the top surface in FIG. 24and which extend from the upper surface to the lower surface. When aningress valve is open, liquid will flow into the chromatography streamthrough an access port from the corresponding ingress channel. Columnaccess ports 436 in this layer of the device are shown with the samenumber label in the top view in FIG. 24.

Referring to FIG. 19, the ingress and egress channels 454 are sealed bya barrier plate 434 having bores 458 aligned to access each of theingress and egress channels and column access bores, and secured by ananchor plate 460 under mechanical pressure. The anchor plate 460 hasbores 462 aligned with the bores 458 and column access bores. Thesebores are shown larger than those in the barrier plate, because thelarger orifice is needed to accommodate conventional connectingfittings. The bores 450 (for example FIGS. 20, 25) are present in thefunctional plates to secure the layers together, and to maintainalignment. The barrier plate 456 is easy to manufacture and ensures aliquid seal between the anchor plate 460 and the ingress and egresschannels 452. However, the anchor plate 460 can be machined withcounterbores presenting a seating surface that may substitute for thebarrier plate 460.

The upper surface of the fluid transfer plate 432 is a differentconfiguration for the left and right valve modules, and it is the onlydifference in structural components for the two modules comprising thecomplete instrument. FIG. 26 shows the configuration of fluid transferchannels 466 for a left oriented module. In this fluid transfer plate464 there are no cross-over channels. The transfer channels 466 aredirected from a column access port at one position in the row of portsto the column access port in the second row of ports immediatelyopposite the first. These structures retain the flexibility ofinterrupted flow having a gap 438 in the channel identical to that inthe right oriented fluid transfer plate. Like the right oriented fluidtransfer module, the left module fluid transfer channels 466 also has aplurality of ingress and egress ports 444 arranged in an array in liquidflow proximity to the transfer channels 466.

The pneumatic valves that define the state of flow in the SMB systemoperate on the same principles as the other embodiments. Pneumaticpressure applied to the diaphragm keeps the valve closed. When pressureis relieved the fluid pressure in the fluid channels in the fluidtransfer plate is sufficient to open them deflecting the diaphragm intothe recesses in the bottom of the pneumatic plate. The offset bores areclose enough to the channels to bridge the gap between the offset boreand the channel to allow liquid to enter or leave the channel. FIG. 27is a plan view of the bottom surface of the pneumatic manifold plate 406for the compact embodiment. The recesses 468 are shown as ovals with themanifold channel 470 depicted as a small circle slightly off center. Therecesses 472 corresponding to the flow intervention gap (see FIG. 27,438) are rotated 90 degrees to be shown as horizontal. This is to beable to maintain the same distance between the ends of the interruptedchannel and the distance between the channel and the offset ingress andegress bores (444 and 446; 466, FIG. 26).

The configuration and inter-relationship of the compact device isfurther elucidated by referring to FIG. 28, a partially explodedcross-sectional left facing view. The optional cover 402, circuit board404, and pneumatic plate 406 are shown as an assembled unit. The halfsolenoid 405 is secured in place via screws onto the circuit board 404.It is important that mounting is secure to prevent leaks at theinterface of the solenoid valve ports and the corresponding bores 424,425 in the pedestal 412. The figure shows that those junctions areflush, and leakage is prevented by applying pressure from the screws tocompress the gaskets sandwiched between the valve ports and thepedestal. The exhaust channel 426 is a three sided open groove open tothe atmosphere on the pedestal 412 and corresponds in alignment to theexhaust port on the underside of the valve body 405.

FIG. 29 is a right facing partially exploded cross-sectional perspectiveview of the valve module components showing the lower surface of thepneumatic manifold plate 406. The pneumatic channel 424 traverses thepneumatic plate 406 from the interior of the solenoid valve body 405terminating in a recess 472. The figure shows one half of the orifice470 within the recess, and the partial lower surface of a left fluidtransfer plate 432 with vertical ingress and egress channels 452.

FIGS. 30 and 31 show a plan view of the right and left configurations ofthe fluid transfer channel 440 and 466 respectively, with the dottedline structures depicting underlying ingress and egress channels 452.There is a corresponding bore at the intersection of the ingress andegress channels 452 to provide a vertical fluid connection therebetween.Valve bodies 405 are shown in their position relative to the liquid flowchannel system.

In actual operation a right and left valve module face each other inopposite orientation with chromatographic columns connecting the twomodules, thereby creating continuous back and forth fluid contact. Aone-way check valve is place in line on each column in order to preventbi-directional flow in the system. Since there is no air in the fluidtransfer system, theoretically, the two modules can be oriented in anyplane. As a practical matter, a horizontal arrangement is mostconvenient for assembling the columns and bleeding air from the columnconnectors. FIG. 32 shows a carriage for horizontally mounting the valvemodules. The carriage comprises a track 473 consisting of foot members474 on which a track member 476 is mounted. Adapter plates 478 areadapted to bolt to the modules. The adapter plates are attached to amovable carrier 482 which slides onto the track member 476 and conformsto the inner female surface thereof. A cam (not shown) is adjusted by acam handle 480, to fix the modules mounted carrier 482 in the desiredposition. This device anchors the modules and permits insertion ofcolumns of varying length.

A more detailed description of a control system 500 for an SMB system inaccordance with the present invention will now be described in moredetail beginning with reference to FIG. 33. Control of an SMB system inaccordance with the present invention to implement a desired processrequires control of the states (open or closed) of the various valvesimplemented in the valve blocks 10 in the accordance with the presentinvention and control of the various pumps 502 that direct the flow offluid in and out of the system. Preferably such control is implementedin a user-friendly manner using a personal computer 504 or similarprogrammable device. Control software implemented on the personalcomputer 504 is used to generate control signals that are provided tocontrol operation of the pumps 502, via an appropriate pumpcommunication adaptor 506, and to control the states of valves in thevalve blocks 10 via appropriate control electronics 508 and valveoperation devices found in the chromatography module 510.

Since the computer processing requirements to implement a control system500 for the present invention are not very demanding, any appropriateconventional personal computer system 504 or similar programmable devicemay be employed to implement the control system 500. Such a personalcomputer 504 should preferably include conventional input and outputdevices, such as a keyboard, mouse, display screen, and the like thatallow a user of the system to interact with the control softwareimplemented therein. Sufficient conventional memory should be providedfor the computer system 504 for storage of the computer program forimplementing the control functions described herein, including thelook-up table to be described below, as well as conventional operatingsystem and other software required or desired for general operation of ageneral purpose personal computer 504. Based on the detailed descriptionand drawing figures, including exemplary screen shot figures, providedherein, a person of ordinary skill in the art of computer programmingfor industrial or laboratory controls will be able to implement asoftware program for performing the user interface and control functionsdescribed herein using conventional programming languages on aconventional personal computer running a conventional operating system(such as Windows or Mac OS).

The control software implemented in the computer system preferablyprovides for control of pump flow rates via a direct communicationbetween the personal computer 504 and the pumps 502 themselves. Forexample, a Universal Serial Bus (USB) cable may be used to connect thepersonal computer 504 to a converter implemented in the pumpcommunication adaptor 506 that takes the USB connection and splits itinto separate serial connections (e.g., in the example shown, fourseparate serial connections, one for each pump. The control software inthe personal computer 504, responding to user input, sends a command toeach pump 502 telling it what flow rate to pump at and the pump 502responds by adjusting its flow rate. Of course, it should be understoodwired or wireless communications between the personal computer 504 andthe pumps 502, and control circuitry therefore, other than thatdescribed by example herein, may be used to control pump operation.

Turning now to FIG. 34. The chromatography module 510 relies on precisecontrol of the valves 512 in the valve blocks 10 (e.g., in the examplebeing presented, a total of 72 valves, 36 on each side of the columns ofthe SMB system in accordance with the present invention, although anyother number of valves may be used as appropriate) to direct the flow offluid in a manner that simulates a moving bed. The starting point forthis control is the control software running in the personal computer504 that generates control signals that are provided, via theappropriate control electronics 508, to implement such precise control.In a preferred embodiment of the present invention, when the controlsoftware needs to alter the current state of the valves 512 within thechromatography module 510, it sends a patterned data stream to anelectronics control board 514 that interprets this control signal inputand directs the action of solenoids 516 (one per valve 512) that, inturn, control the gas or other fluid pressure applied to the diaphragm14 in the valve block 10 for each valve, thereby opening or closing theappropriate valves in the manner discussed above.

For example, the state (open or closed) of each valve 512 in the systemmay be defined by the bits in a patterned bit stream that is generatedby the control software running in the personal computer 504 andprovided at the control electronics 508 via a USB or other appropriatewired or wireless connection 518. A USB interface chip 520 on the board514 takes in the digital input, processes it, and turns on and off wiresleading from the interface chip 520 to devices, e.g., a series of shiftregisters 522, that store and present the individual control bits foroperation of each valve solenoid 516. One of the wires from theinterface chip 520 is connected to the first of the series connectedshift registers 522 for carrying the bit pattern corresponding to thedesired state of the valves 512 to the shift registers. This bit patternis fed into the series connected shift registers 522 to load the shiftregisters as necessary to control the desired number of valves 512. Forexample, if eight bit shift registers 522 are used, each shift registercan hold and present the bits defining the state of eight valves 512. Tocontrol 36 valves, for example, eight bits are first shifted into thefirst shift register 522. These eight bits are then shifted to thesecond shift register 522 in series as eight additional bits are shiftedinto the first shift register 522. This process is repeated, shiftingthe bit stream downward in the series connected shift registers untilsufficient shift registers are loaded with the desired bit streamrepresenting the desired state for 36 valves. Timing and control of thisbit shifting/loading process is provided by another wire leading fromthe interface chip 520 to each shift register 522, which acts as aserial clock.

After the appropriate bits have been set in the shift registers 522 anupdate of the state of the valves 512 is triggered by a third wireleading from the interface chip 520 to each of the shift registers 522.This third wire is connected to the shift register pin for presentingthe new loaded shift register contents at the specified output pins ofeach shift register 522. These shift register output pins are connectedto appropriate power transistors 524 which are sufficient to driveenough current to turn on or off a connected solenoid 516 depending uponthe bit value (0 or 1) presented to its input by the shift register. Asdiscussed above, each solenoid controls the delivery of high pressuregas or other fluid to the membranes 13 in the valve blocks 10 to controlthe valve state (on or off). Thus, the digital outputs from the shiftregisters 522 direct the turning on or off of each valve in thechromatography module 510.

In the exemplary embodiment being discussed herein, two control boards514 are used to control 72 valves 512 in the system (each board 514controls 36 valves). The interface chip 520 on the first board 514 sendshalf of the control bit stream to its series of shift registers 522 andthe other half is relayed to the second control board 514 throughanother wired connection. (Thus there need not be a separate USBinterface on the second control board.) If more valves need to be addedto the device, the overflow from the last shift register on each boardmay be daisy-chained to yet another board of the same configuration.

The control software running on the personal computer 504 preferablyprovides a user-friendly interface that allows a system operator todefine the state (open or closed) of the valves 512 in the system.Preferably the software provides for both manual control of the valvestate across the module and an automated switching of those states.

Manual control provides the ability, for example, to test thechromatography module 510 to determine if its individual components arefunctioning properly. Manual control of the valve states may beaccomplished by the user activating the control software and opening agraphical user interface window that depicts the current valve state ofthe module. An exemplary graphical user interface 528 of this type isillustrated in FIG. 35. This manual control user interface 528 presentsa check box type control interface 530 for each valve in the system thatmay be controlled. The user selects the desired valve state for eachvalve by checking or un-checking, as appropriate, the check box 530corresponding to the valve. Once the desired valve states are selectedin this manner the user clicks an update valve configuration button 532that commands the software to update the state of the valves as justdefined.

After the user clicks the update valve configuration button 522 thesoftware processes the desired valve state by checking each valvedepicted in the user interface 528 against an internal look up tablethat indicates which location on the electronic control board shiftregisters 522 corresponds to that valve. Once the location of each valvehas been identified, the software creates the appropriate sequence ofbits (1s and 0s) that are loaded onto the shift registers 522 in themanner described above. Finally, the controlling electronics triggersthe shift registers 522 to present the loaded bits at the shift registeroutputs to set the valves across the system to their new state, in themanner described above.

To direct the controlled switching of valve states in an automatedmanner, e.g., to perform a separation, the controller softwarepreferably provides the user the ability to input sequences of valvestates in the form of scripts that the software runs in an automatedfashion. An exemplary graphical user interface showing an exemplary userdefinable control script 514 of this type is shown in the screen shotillustration of FIG. 36. These scripts consist of a series of steps in aprocess, each of which defines the system valve states in a mannersimilar to that used to define the valve states in the manual controlmethod described above. However, in this case each step also includes auser selected time period, e.g., in seconds, that the system will remainin the defined valve state configuration before switching to the nextstep. The graphical user interface preferably provides virtual buttonsfor allowing a user to add 542 or delete 544 steps from the script. Ifthe user selects to add 542 a step a new graphical user interface 546window, as shown by example in FIG. 37, may pop up to allow the usereasily to define the state for each valve for that step.

Returning to FIG. 36, upon the user selecting a button 548 to run adefined script, the first step in the script is read and processed, in amanner similar to that described above for the manual control, togenerate a bit pattern that is sent to the control electronics 508. Thesoftware waits for the time duration specified by the step, then repeatsthe process for the valve states defined for the second step. When theend of the scripts has been reached, the software can either loop backto the first step, at the users selection 550, or terminate execution,based on a configuration setting for the step.

It should be understood that the control software may provide additionaland/or different graphical user interface options, and/or in a differentformat, from that presented by example herein. For example, thegraphical user interfaces may provide options for the user to saveindividual valve state configurations and/or scripts and retrieve themfor later use.

The principle of standard affinity chromatography relies upon the highspecificity of the binding event of the target protein to theligand-bound resin. In a typical batch procedure, target protein presentin a feedstock contacted with the resin is immobilized on the resin. Thecolumn is then thoroughly rinsed with a buffer of the same or similartype as that in the feedstock to remove all the unbound contaminants.Finally, the target protein is eluted with an affinity neutralizingagent, and the target is released from the resin and collected. It isgenerally accepted by those skilled in the art, that it is desirable totake full advantage of the binding specificity by binding underconditions that achieve maximum static immobilization of the targetprotein to the resin, i.e. under conditions in which the feedstockcontains none or very low concentrations of the affinity releasingagent.

However, this strategy has its trade-offs. Most complex protein mixturescontaining the target protein of interest also contain unrelatedproteins that have weak binding affinity for the column resin,presumably because they share or mimic binding domains. When theaffinity releasing agent is applied to the column these contaminatingproteins unrelated to the target are also released and contaminate thefinal eluant. If higher purity is desired, the target protein must bere-purified using another separation technique which will lead to areduction in yield.

Applicants have discovered that higher levels of purity of a targetprotein can be achieved under conditions in which the binding affinityis attenuated and one concentration of affinity releasing agent is usedthroughout the entire standard SMB run. These conditions are termedisocratic. While not being bound to any particular theory, it appearsthat at concentration in a range of less than that required to releasebound protein from the resin, the target protein migrates in a mobilefront, but, because of residual affinity interactions with the affinityligand, is retarded in its passage through the column in contrast toproteins having little or no affinity for the resin. A higher attainedlevel of purity in the extract is possibly explained by considering thatthe weakly bound contaminating proteins found in conventional affinitychromatography are not retarded in elution and pass along with the bulkof unrelated proteins into the raffinate.

This phenomenon also may be accounted for by the nature ofcountercurrent flow in SMB chromatography, since flow rates and columnswitching times can be varied to sharpen the target peak, and reduce theoverlap of extract and eluent. A further enhancement of purity can beobtained by “peak shaving” by adjusting flow rates and valve switchingto permit the leading edge of the extract peak to be sacrificed in theraffinate. While theoretical models can be constructed to predict theselection of optimum running conditions, empirical observation isefficacious and does not require undue experimentation. This isillustrated in Example 2 hereinbelow. In this example, experiments arepresented using a his-tagged recombinant protein having a selectiveaffinity for chelated nickel bound to a resin matrix. Many other modelaffinity systems are known, to which the present SMB chromatographydevice and methods are applicable, and include both recombinant tags andendogenous affinity domains exhibiting selective specificity for aligand bound to a column matrix. One particularly important affinitysystem is the purification of antibodies and other molecules havingimmune specificity. Among the most prevalent methods are affinitysystems exploiting the affinity domains contained in IgG specific forProteins S and G. According to the method of the invention, the skilledartesan would select pH buffer systems at a pH below the binding pH (pH7.4-9.0), testing a few pH point between pH 3.0 and pH 7.4 to determinea range of pH where the antibodies are retarded in mobile phase relativeto the other contaminating proteins in a crude acites or other extract.

The simulation of a moving bed in the present invention requirescoordinated opening and closing of diaphragm valves during operation,modulation of valve switch times and fluid flow rates, and the abilityto collect desired fractions of raffinate and eluate. The process iscomputer controlled and automated through a standard personal computerinterfaced with the SMB unit. A software program governs the pneumaticcontroller that operates the solenoids which activate individualpneumatic lines, thus controlling opening and closing of individualdiaphragm valves. A software program compatible with multiple operatingsystems (Windows, Linux, or Macintosh) programmed in C++ language ispreferred. FIG. 14 gives a simplified diagram of the control system. Acomputer 301 interfaces a micro controller digital I/O 302, whichconverts computer signal to pneumatic valve driver 303 instructions. Thelatter controls operation of individual valves 304 which operate in avalve 10 as previously illustrated in FIG. 3.

Example 1

A valve block of the type depicted in FIGS. 4A and 4B was tested todetermine the pressure differentials needed for proper opening andclosing of the diaphragm ports without causing any leaks. Gas pressurewas controlled manually for each of the four nipples (FIG. 3, 42) Inletfluid pressure was also controlled manually and liquid flow rates weremeasured as a function of pressure differential between the pneumaticand fluid inputs (pneumatic >fluid). A valve inlet 9 transfers a liquidto the linear common groove (FIG. 3, 46). The other outlets are referredto from left to right as 5, 6, 7, 8, and 10′ in FIG. 15.

The experiments were operated at pressure differentials up to 20 psi.The data shows that pressure differentials of 1 psi or less wereadequate to seal the valve ports at flow rates expected for the SMBsystem (1-4 mls/min.). Such pressures prevented any leaks from appearingat one minute intervals. Flows were observed for all output ports. Itwas further observed that back pressure from connection of up to fourchromatographic columns did not interfere with flows or cause pressurestress on the system.

Example 2

To evaluate continuous isocratic SMB chromatography in purifying atarget affinity-tagged protein, a series of experiments were devised andcarried out. A pET-EKLIC plasmid encoding his(6)-tagged annexin I (43Kda) was transformed into BL21 DE3 host cells and target proteinexpressed using TB autoinduction medium as described in Novagen UserProtocol TB383 Rev. H 1005, based on a method summarized by F. WilliamStudier, Protein Expression and Purification, 41: 207 (2005). Cells wereharvested after overnight incubation at 30 degrees and soluble extractsprepared using BugBuster™, Benzonase®, and rLysozyme (Novagen) accordingto standard protocols. Crude extracts (16 ml) from 1 gram cell pelletswere adjusted to the desired imidazole concentrations and used forpurification on Novagen Ni-MAC columns in the SMB device of the presentinvention according to step batch conventional affinity chromatographyor continuous isocratic SMB. Running buffers also contained 50 mM sodiumphosphate pH 8.0 and 300 mM sodium chloride.

To establish isocratic conditions for the continuous isocratic SMBexperiments, separate runs were made at isocratic concentrations of 115mM imidazole, 150 mM, 175 mM, and 250 mM. As expected, all of the targetprotein was recovered in the raffinate at 250 mM imidazole, a knownelution concentration. Elution of target into the raffinate was alsofound at 175 mM. However, at 115 mM and 150 mM, recovery of targetprotein in good purity and yield was obtained.

The column fractions collected at the raffinate and eluate ports duringexperimental affinity step SMB or isocratic SMB runs were analyzed bySDS-PAGE (10-10% gradient gels) and Coumassie Blue staining. Eluatelanes were loaded with approximately 3 μg sample, and raffinate laneswere loaded with approximately 20 μl sample. The results are depicted inFIGS. 18A and 18B. Protein for the various fractions was quantified byBCA assay and spectrophotometrically. Purity was assessed by scanningdensitometry. A summary is given in the Table below. The table includesdata for a second isocratic run in which the valve switching times werereduced from 2.0 minutes to 1.5 minutes. Data were also compared to asingle column batch run (gel not shown). TABLE IMAC Mode Yield (mg)Recovery Target Purity Isocratic 2 min. switch 40  87% 98% Isocratic 1.5minute 34  73% 96% Step affinity SMB 51 100% 89% Single column batch 3.3mg/ml 100% 81%

In these experiments the isocratic SMB mode produced greater purity thanthe step affinity mode, which in turn produced greater purity than thesingle column batch mode. There is some sacrifice of yield when higherpurity is sought. The experiments also demonstrate how altering theparameters of the SMB can affect the result. Other advantages of thepresent invention will be apparent to those skilled in the art.

1. A valve module comprising: a pneumatic plate having a top surface anda bottom surface, said top surface having a plurality of pneumatic boresextending from a pneumatic valve port on said upper surface andterminating in recesses on said pneumatic plate bottom surface; a fluidtransfer plate having an upper and a lower surface, said upper surfacehaving a plurality of fluid transfer channels, access ports, and anarray of offset ports in liquid flow proximity to said fluid transferchannels and aligned with said pneumatic bottom surface recesses, and abottom surface having a plurality of ingress and egress channels withbores communicating between said offset ports and said ingress andegress channels; a valve diaphragm comprised of a pliant pressureresponsive film disposed between said pneumatic plate bottom surface andsaid fluid transfer plate upper surface; and an anchor plate havingaccess ports and fluid ingress and egress bores in alignment with saidbarrier plate corresponding bores.
 2. A compact valve module comprisinga pneumatic plate comprising a having a top surface, a bottom surface,and a core, said top surface having a plurality of elongated pedestalsproviding seating surfaces for a plurality of solenoid valves, with saidupper pedestal surface containing a plurality of pneumatic boresextending from a pneumatic valve port on said upper pedestal surface andterminating in recesses on said pneumatic plate bottom surface, a secondbore extending from a pneumatic manifold port on said upper pedestalsurface to communicate with a transverse manifold channel embedded insaid core, and an exhaust channel contiguous to said pedestals, saidbores and exhaust channel being in alignment with corresponding ports ofsaid solenoid valves so seated on said circuit board an electroniccircuit board bearing electrical receptacles for powering said solenoidvalves a fluid transfer plate having an upper surface and a lowersurface, said upper surface having a plurality of fluid transferchannels etched thereon terminating at each end of said channel invertical column port bores arranged in two rows along opposite sides ofsaid plate, and a series of offset bores in liquid flow proximity tosaid fluid transfer channels aligned with said lower pneumatic platesurface recesses, said offset bores extending through said fluidtransfer plate to communicate with four or more substantially linearfluid ingress and egress channels etched on said transfer plate lowersurface a valve diaphragm disposed between said pneumatic plate bottomsurface and said fluid transfer plate upper surface; and an anchor platecontaining column communicating bores in corresponding verticalalignment to said column access bores and said fluid transfer platebores.
 3. An annular valve block module integral to a simulated movingbed chromatography device for affinity purification of biologicalsubstances comprising a pressure manifold having a top surface and abottom surface containing a plurality of bores arranged radiallyextending perpendicularly from said top surface and terminating inrecesses on said bottom surface a fluid transfer plate having an upperand a lower surface having a plurality of radial U-shaped interruptedgrooves etched thereon facing to the perimeter of said upper surfaceterminating on their ends in on a first and second column bore insubstantially central alignment to a recessed bore of said pressuremanifold proximity to said U-shaped interrupted groove, and furtherhaving a series offset bores arranged spacedly to said grooves alignedsubstantially centrally with said recesses of said manifold, said boresextending through said fluid transfer plate to communicate with aplurality of concentric or semi-concentric fluid ingress or egresschannels etched on said fluid transfer lower plate surface, said boresand channel being further characterized in arranging all said borescommunicated with egress channels being on the upstream segment of saidU-shaped grooves and all the said ingress bores being arranged on thedownstream segment of said grooves a cutoff valve positioned betweensaid ingress and egress offset bores to interrupt flow in the U-shapedchannels between said ingress and egress bore arrangements a valvediaphragm comprised of a pliant pressure responsive polymer disposedbetween said pressure manifold bottom surface and said fluid plate uppersurface a barrier membrane forming a sealing surface at said lowersurface of said transfer plate whereby to form a lower barrier wall ofsaid circular or semi-circular channels and having column access portsto communicate with aligned chromatographic columns and said fluidingress and egress channels an anchor plate having an upper and a lowersurface containing column communicating ports of said barrier membranean optional sealing layer to prevent leaks between the anchor plate andthe lower surface of said lower surface of said transfer plate an accessport for each liquid ingress and egress channel comprising a boreextending perpendicularly from the lower surface of said anchor platethrough said barrier membrane to communicate with each fluid ingress andegress channel.
 4. A method of purification of biological or chemicalsubstances by the device of claims 1 having four zones of one or morechromatographic columns, namely, adsorption, wash, elution, andregeneration, a downstream propelled flow of fluid, and a cycling ofvalve positions in recurrent stepwise configuration comprising applyingto a first zone a feedstock containing a target substance of interest tosaid adsorption zone under conditions allowing said target substance tobind to a solid or semi-solid matrix having a chemical moiety with anaffinity to said target allowing a portion of unbound contaminants toexit via a raffinate port in said downstream fluid flow, whilepreventing the flow of fluid into said downstream zone by closingappropriate valves according to claim 1 switching the valve positions toadvance said adsorption zone, one column at a time, to the wash zone,wherein contaminants are allowed to exit via an effluent port, whilepreventing the flow of fluid into said downstream zone by closingappropriate valves according to claim 1 switching the valve positions toadvance said wash zone, one column at a time, to the elution zone,wherein said target substance is dissociated from said solid orsemi-solid matrix by adjusting the fluid conditions and is allowed toexit via a product port, while preventing the flow of fluid into saiddownstream zone by closing appropriate valves according to claim 1switching the valve positions to advance said elution zone, one columnat a time, to the regeneration zone, wherein the conditions required forbinding said target substance to said solid or semi-solid matrix arere-established, and fluid is allowed to exit via an effluent port, whilepreventing the flow of fluid into said downstream zone by closingappropriate valves according to claim 1; and repeating sequentially thesame configuration of valve positions advanced one column at a time. 5.A method for isocratically purifying a target protein having endogenousselective affinity for a ligand from a mixture of proteins in afeedstock using a simulated moving bed chromatography device having aplurality of chromatography columns substantially filled withligand-bound resin, and which has inlets for feedstock and eluent andoutlets for extract and raffinate comprising selecting a concentrationof an affinity neutralizing agent sufficient to prevent static bindingof said target to said ligand-bound resin while selectively retardingpassage of said target protein through said ligand-bound resin in amobile phase in contrast to proteins in the mixture not having suchaffinity adjusting the concentration of affinity neutralizing agent inthe feedstock and eluent to the selected concentration; and processingthe feedstock through the simulated moving bed chromatography device toobtain purified target from said extract outlet.
 6. The method of claim4 wherein the target protein is a molecule having immune specificity.